Ribonucleic acids (RNA) are biopolymers contained in cells of all living organisms and in some viruses, which play various roles, primarily conveying the hereditary information of DNA, in constructing proteins. RNA is thus employed in investigations of the genome under microarray-hybridization experiments and RT-PCR, as well as, for example, northern blots, RNase-protection assays, or cDNA-syntheses. The results of such experiments, and the significances of their results, are largely dependent upon the qualities of the RNA samples employed.
The size distributions of RNA biopolymers are measures of the integrity, and thus the qualities, of the RNA samples involved. RNA size distributions vary with the origin of the material involved and the method by which it was prepared, but are significantly affected by contamination by RNA degrading enzymes (RNases) or mechanical shearing forces due to improper handling. In any event, a shift in the lengths of RNA polymers to shorter lengths is observed whenever degradation has occurred.
Traditionally, RNA biopolymers are analyzed by gel electrophoresis. Lab-on-a-chip analyses using the Agilent 2100 Bioanalyzer, as provided by the applicant Agilent Technologies, provide an accurately reproducible, high-resolution, approach to gel-electrophoresis. The Bioanalyzer has become the industry's standard, in particular for RNA analysis. Digital electropherograms obtained using it thus represent ideal starting points for more thorough analyses.
Quality of an RNA molecule can be understood as a measure for its integrity. For example, an RNA sample will exhibit a high degree of integrity, if its molecules have survived extraction from cells or tissues as a whole without suffering degradation or breakage due to shearing forces. A measure for the integrity can indicate e.g. to what extent the sample exhibits signs of degradation or shearing, for example with the highest values displayed by samples that have remained intact during preparation.
Reliable quality determinations have thus far been obtainable using manual, and therefore more or less subjective, methods only. Under manual methods, each individual sample must be visually inspected for integrity by an experienced biochemist before it may be employed in subsequent experiments. Manual quality determinations have become unacceptable as the number of RNA experiments to be performed grew and high-throughput analytical methods appeared.
Methods that will allow estimating the qualities of RNA samples based on a single feature, or only a few features, are known. The best approach that has emerged to date establishes a criterion for the ratio of the areas under the curves for the 28S-rRNA-fragment and the 18S-rRNA-fragment. Theoretically, intact RNA samples will have a ratio of 2. This ratio decreases as degradation progresses, and yields an initial, but relatively inaccurate, means for distinguishing between intact and degraded RNA, since large deviations of this ratio from the theoretical value frequently occur in practice. This criterion is regarded as the state of the art in automatic determination of the qualities of RNA samples.